Phosphorylation of Inositol 1,4,5–Trisphosphate Analogues by 3‐Kinase and Dephosphorylation of Inositol 1,3,4,5‐Tetrakisphosphate Analogues by 5‐Phosphatase

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Abstract
A series of 32P‐labeled d‐myo ‐inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] analogues was enzymically prepared from the corresponding d‐myo–inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] analogues using recombinant rat brain Ins(1,4,5)P3 3‐kinase and [γ‐32P]ATP. Ins(1,4,5)P3 analogues with bulky groups at the 2‐OH position, substitutions of phosphates by thiophosphates and d‐6‐deoxy‐myo ‐Ins(1,4,5)P3 were tested. Using [3H]Ins(1,4,5)P3 and ATPγS, a [3H]Ins(1,3,4,5)P4 analogue with a thiophosphate at the D‐3 position was prepared. The D‐4 and/or D‐5 phosphate group seemed to be important for 3‐kinase activity, while the OH group at position 6 was not crucial. The addition of bulky groups at the 2‐OH position did not prevent phosphorylation.The labeled Ins(1,3,4,5)P4 analogues were purified and their degradation by type‐I Ins(1,4,5)P3/Ins(1,3,4,5)P4 5‐phosphatase was compared with the degradation of Ins(1,3,4,5)P4. Substitution of the phosphate group at positions 1 or 3 by a thiophosphate, or the addition of bulky groups at the 2‐OH position did not prevent degradation. d‐6–Deoxy‐myo ‐inositol 1,3,4,5‐tetrakisphosphate could not be degraded by the 5‐phosphatase, indicating the importance of the 6‐OH group for 5‐phosphatase action. d‐6‐Deoxy‐myo–inositol 1,3,4,5‐tetrakisphosphate could be an important tool in elucidating the cellular functions of Ins(1,3,4,5)P4

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